Replicative DNA helicases provide a critical function in DNA metabolism as they separate the 2 strands of the parental DNA in preparation for chromosome duplication. In eukaryotic cells, a hexameric ATPase called the MCM2-7 complex is an excellent candidate for the replicative DNA helicase, but little is known about how it unwinds DNA in the context of DNA replication. To study the biochemical mechanism of the replicative DNA helicase, a cell-free system derived from Xenopus laevis eggs is being used that recapitulates efficient, cell-cycle regulated DNA replication in a soluble protein environment. In this system, when DNA replication is initiated in the presence of DNA a polymerase inhibitor, many kilobasepairs of DNA are unwound by a highly processive DNA helicase in the absence of detectable DNA synthesis. This "hyper-unwinding" is dependent on MCM2-7 and the replication factor Cdc45, indicating it reflects functional uncoupling of the replicative DNA helicase from the polymerase. The hyperunwinding assay will be used to study the biochemical properties of the helicase in its native chromosomal context. In Specific Aim 1, chromatin immunoprecipitation will be used to determine which of the known DNA replication proteins are physically associated with the uncoupled helicase. In Specific Aim 2, proteins other than MCM2-7 and Cdc45 that are required for DNA unwinding by the helicase will be identified. In Specific Aim 3, the mechanism by which Cdc45 stimulates helicase activity will be studied. In Specific Aim 4, we address whether MCM2-7 unwinds DNA by translocating along single-stranded or double-stranded DNA. These studies are expected to yield important insights into the molecular mechanisms underlying DNA replication. While mutations that severely inhibit the function of the replicative DNA helicase will cause lethality, subtle mutations might cause genomic instability, and ultimately, cancer. In addition, this helicase is an attractive target for chemotherapy. Therefore, understanding the enzymology of this important enzyme is potentially of great interest for the treatment of human disease.